Thermostable varicella zoster virus

ABSTRACT

PCT No. PCT/US94/14722 Sec. 371 Date May 22, 1996 Sec. 102(e) Date May 22, 1996 PCT Filed Dec. 19, 1994 PCT Pub. No. WO95/17503 PCT Pub. Date Jun. 29, 1995A thermostable varicella zoster virus (tVZV) is useful for the preparation of a vaccine against chickenpox. The tVZV was selected from a population of virus which survived stringent heat inactivation conditions. The surviving virus is used to provide seed virus to produce a new vaccine with enhanced stability.

CROSS-RELATED TO OTHER APPLICATIONS

This application is a 371 of PCT/US94/14722, filed Dec. 19, 1994, whichis a continuation of U.S. application Ser. No. 08/171,048, filed Dec.21, 1993, abandoned.

BACKGROUND OF THE INVENTION

This invention is concerned with the provision of a thermostablevaricella virus for vaccine production. Varicella zoster virus (VZV)causes chicken-pox and zoster (shingles). Chickenpox is a highlycontagious disease that occurs in persons with no VZV immunity. Morethan 90% of the population is exposed during the first two decades oflife. The disease is a severe threat to the immunosuppressed and toadults. In many cases, VZV becomes latent in dorsal root ganglion cells.Shingles, a painful chronic condition, occurs when VZV is reactivatedfrom the latent state.

Prevention of chickenpox by vaccination is a desirable goal, and theinstitution of universal childhood vaccination with a live attenuatedvaricella vaccine is envisioned. The prior art has reported thepropagation of VZV in various cell culture systems and the use of live,attenuated, cell-free VZV as a vaccine. U.S. Pat. No. 3,985,615describes the production in guinea pig primary embryonic cells ofattenuated varicella virus. Virus produced according to that process,the Oka strain of VZV, is suitable for vaccine use and has beendeposited with the ATCC as VR-795, although other strains of varicellamay be used to produce attenuated VZV according to the U.S. Pat. No.3,985,615 and other known processes (see U.S. Pat. Nos. 5,024,836; and4,000,256). U.S. Pat. No. 4,008,317 describes the cultivation of atemperature-sensitive mutant of VZV in WI-38 cells. Compositions usefulfor the maintenance of viable VZV, such as SPGA, are also known in theart, (see U.S. Pat. Nos. 4,147,772; 4,000,256; 4,337,242, and4,338,335).

VZV is a member of the herpesvirus family. VZV has been isolated andprovided as a live attenuated virus vaccine which is effective toprevent varicella infection in children (U.S. Pat. Nos. 3,985,615;4,000,256; 5,024,836). No effective, inactivated VZV vaccine has beendeveloped, and VZV rapidly loses viability at ambient temperatures.Thus, a constant problem with VZV vaccines of the past has been the needto store the virus at temperatures below freezing point. This hastypically meant that the live attenuated vaccine, even when lyophilized,must be stored in a stabilizing medium at reduced temperatures, such as-15° C. or -20° C. Under these conditions, the live attenuated vaccineviability half-life is approximately 36 months. At -70° C., the haftlife is much longer (on the order of many years). However, where thelive attenuated vaccine must be stored at higher temperatures, such asat 4° C. or higher, as in third world countries where refrigeration ofvaccines at any temperature is difficult, the virus viability drops offvery rapidly. Thus, there has been a need for a more stable liveattenuated VZV vaccine. This invention meets that need.

SUMMARY OF THE INVENTION

A thermostable live attenuated varicella zoster virus (tVZV) is producedby selection and growth of virus which survives heat inactivation. Itwas not predictable that heat stable VZV would be produced. The tVZV isuseful to produce a new live attenuated varicella zoster virus vaccinewith innately increased thermostability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Linear regression curve showing thermostability of the new tVZVof this invention at 35° C.

DETAILED DESCRIPTION OF THE INVENTION

Varicella zoster virus (VZV) can be isolated from the papular eruptionsof children in the acute phase of chickenpox. VZV isolated in thismanner is cultured in vitro over multiple passages to produce a live,attenuated virus. This can be accomplished according to U.S. Pat. No.3,985,615, hereby incorporated by reference.

According to one embodiment of the instant invention, a live attenuatedVZV vaccine is lyophilized after culture and attenuation according tomethods known in the art. The lyophilized virus is then subjected to anextended period, about 5-19 days, at an elevated temperature, 25°-75° C.Following the inactivation cycle, residual viable virus is recovered andselected in tissue culture. The virus selected in this manner is thenpropagated and formulated, either in a liquid or lyophilized state,according to methods known in the art, and is provided as a liveattenuated VZV vaccine of improved thermostability, referred tohereinafter as tVZV.

The utility of attenuated, cell-free VZV as a vaccine to preventchickenpox has been demonstrated. Multiple clinical studies haveconclusively proven this utility, and such proof is now part of theprior art see for example, Pediatrics, 88 (3), 604-607 (1991);Pediatrics, 87, (5), 604-610 (1991)!. Thus, the tremendous contributionthat this invention makes to the art is that it provides a thermostableVZV, tVZV, making ready availability of the virus for stable vaccineformulation more feasible. It was not predictable that a heat stable VZVwould be isolated.

tVZV prepared according to this invention may be formulated as a vaccineaccording to methods known in the art, and administered according toregimens by now well established. For example, the live, attenuated,cell-free tVZV product of this invention may be diluted into stabilizer,filled in bottles, lyophilized in unit doses such that upon storage atabout 4° C. or lower, and preferably at about -20° C., a dose of about1000 PFU will be available at the time of use. The VZV vaccine producedaccording to the process of this invention may be used in unit doseformulations to inoculate humans to induce immune-responses protectiveagainst infection by virulent strains of VZV. Preferably, at a minimum,a dose of about 2000 PFU/ml (1000 PFU/0.5 mL dose) is administeredsubcutaneously or intramuscularly. Doses of attenuated VZV as high as atotal of 15,000 to 20,000 PFU have been administered and are acceptable.

The tVZV of this invention is useful to prepare a thermostable liveattenuated VZV vaccine. It is also useful to prepare an immunogen toboost immune responses to VZV in seropositive individuals. The tVZV isalso useful as a tool to analyze genomic changes responsible inconferring increased thermostability to viruses. Accordingly, the tVZVgenome of this invention is analyzed for restriction fragment lengthpolymorphisms (RFLP's) or the genome is sequenced according to methodswell known in the art. In this manner, variations in the genomeconferring this enhanced thermostability are identified.

Vaccine formulations of VZV are well known in the art. Thus, accordingto U.S. Pat. No. 3,985,615, the VZV may be formulated in a sterile, 5%sucrose solution. More complex formulations, such as SPGA (0.218Msucrose, 0.00376M KH₂ PO₄, 0.0071M K₂ HPO4, 0.0049M potassium or sodiumglutamate, 1% human serum albumin) which further enhance the stabilityof the VZV are also known in the art. Thus, U.S. Pat. No. 4,000,256discloses a stabilizer comprising sucrose, albumin, glutamate, andphosphate. Similarly, U.S. Pat. No. 5,024,836 discloses a lyophilizedcomposition comprising VZV and measles, mumps, and rubella in astabilizer wherein the moisture content is maintained between 2-8% toimprove virus stability. These formulations are referred to genericallyherein as pharmaceutically acceptable carriers and the disclosure ofthese patents is hereby incorporated by reference.

In addition to the tVZV and a pharmaceutically acceptable carrier,combination vaccines may be prepared incorporating other vaccineentities. Thus, a tetravalent vaccine also including measles, mumps andrubella (M-M-R-V) is contemplated by this invention. Such combinationsusing attenuated but not thermostable VZV are known in the art seeD'Hondt, EP-A-0 252 059; see also Arbeter, et al., Pediatrics,76(suppl.):742-747 (1986)!. tVZV could likewise be combined with knownDTP vaccines, influenza, hepatitis B, hepatitis A, Haemophilusinfluenzae b polysaccharide vaccine, Streptococcus pneumoniaepolysaccharide vaccines.

These formulations as well as other known methods of formulating andstoring VZV are applicable to the production of a vaccine incorporatingthe tVZV of this invention.

Thus, this invention provides, in one embodiment, a process forpreparing a live attenuated thermostable varicella zoster virus (tVZV)having improved stability, which comprises:

a) heating a lyophilized preparation of live attenuated VZV under highlyinactivating conditions; and

b) selecting and culturing residual live VZV. As defined herein, "highlyinactivating conditions" refers to a heat treatment in which anywherebetween 1/100 to 1/1000,000 virus particles in a given preparationsurvive in a viable state. Preferably, an inactivation with a survivalrate of about 1/1500 is used.

In another embodiment, this invention provides a thermostable varicellazoster virus having a rate of inactivation equal to about 50% per hourat 35° C., as compared with a rate of 79% per hour for untreated VZVunder the same conditions. Thus, according to this method, a decrease inthe rate of inactivation at 35° C. of about 38% (50/79×100) for a givenstrain of VZV can be expected based on the instant patent disclosure. Inaddition, from this invention, it is predictable that at least aproportional increase in half life, from about 36 months to about 60months, occurs when the virus is stored lyophilized at -15° C. or below.Further enhancements in thermostability are achievable by processing thethermostable VZV of this invention through additional heat inactivationand selection of a yet more thermostable VZV isolate. The 38% enhancedstability of tVZV demonstrated herein at 35° C. is predictably at leastproportionally applicable to tVZV stored at reduced temperatures such as4° C., -20° C., or -40° C. Furthermore, optimization of stabilizingsolutions or storage conditions, such as the use of 2-4% moisture in alyophilized formulation, according to the teaching of U.S. Pat. No.5,024,836, hereby incorporated by reference, may be applied to furtherenhance the stability of the tVZV of this invention.

In another embodiment, this invention provides a method of using a tVZVto make an anti-VZV vaccine which comprises providing a minimum of about1000 plaque forming units of the tVZV in a pharmaceutically acceptablecarrier.

Another embodiment is a vaccine comprising the tVZV.

The infectivity titers of varicella zoster virus (VZV) preparations areobtained by an agarose-overlay or liquid overlay procedure described byKrah et al., (J. Virol. Methods, 1990, 27:319-326). Briefly, this methodinvolves culturing MRC-5 cells, which are susceptible to VZV infection,to an actively replicating state, that is, to a point where the cellsare about 50-80% confluent. Virus is then overlaid onto the cellmonolayer in a minimal volume, allowed to attach to the cells and thenadditional growth medium is added. After several days of growth, thecells are exposed to a protein stain, and clear areas, plaques, arecounted. Thus, for a known volume of viral inoculum, the number ofplaque forming traits (PFU) per milliliter represents a good measure ofvirus yield. Multiplied by the total volume of cell-free virus obtainedfrom any given viral preparation, the total number of PFU may becalculated.

tVZV prepared according to the method of this invention has beendeposited with the ATCC under the Budapest Treaty on Nov. 15, 1993 andaccorded deposit numbers of: VR 2437, VR 2438, and VR 2439.

The foregoing description teaches generally how to make the VZV vaccineof this invention having improved thermostability. The followingexamples are provided to teach more particularly how this invention maybe carried out. However, the invention should not be construed as beinglimited to the specifics of the examples.

EXAMPLE 1

Thermal Inactivation and Recovery of Thermostable VZV

Five hundred intact, unreconstituted vials of Varivax®, lot CR453,obtained from Merck & Co., Inc., were heated at 50° C. for 12 days. All500 heated vials were placed at -20° C. storage pending analysis.Subsequent analysis gave the following:

1. Residual live virus was titered at 2.4 PFU/ml, while the standard,unheated CR453 vials were titrated at 3830 PFU/ml. Thus, there was a99.94% reduction in viable VZV under these conditions of thermalinactivation. Thus, a treatment analogous to this is defined herein as"highly inactivating conditions".

2. Total VZV antigen by dot-blot assay was 9.8 units/ml, in the heatedvaccine. Standard unheated vaccine tested simultaneously gave 9.4units/ml. The value at time of original release in 1987 was estimated at9.5 units/ml.

3. Western Blot: This assay indicated no grossly detectable differencesin heated and unheated CR453. Both human zoster serum, (a polyclonalanti-VZV serum), and mouse monoclonal antibody to virus glycoprotein 1were used.

EXAMPLE 2

Demonstration of Enhanced Stability of Varicella Recovered from HeatTreatment

Four vials of heat inactivated CR453 prepared as described in Example 1,and one vial of normal CR453 were each reconstituted with 0.7 ml steriledistilled water and placed on ice. From each heat inactivated vial, 0.5ml (estimated 1.2 plaque-forming units) and 0.5 ml from the unheatedvial (estimated 1915 plaque forming units) was used to inoculate MRC-5cells in culture, at 0.1 mL per 60 mm plate, five plates per isolate.The medium used was EMEM containing 2% fetal calf serum plus neomycinand 2 mM Glutamine.

It was anticipated, based on the known titer of 0.24 PFU/0.1 mL, thatunder the conditions described above, each plate inoculated with heatedVZV would receive one or less live VZV plaque forming units.

These samples were taken through 5 passages as summarized in Table 1below, essentially as follows:

Passage level 1, P1

Prior to inoculation at each level of virus passage, MRC-5 cells weregrown in culture to near confluence. For P1, the medium was aspiratedfrom cell culture sets (one set for each VZV isolate) consisting of five60 mm plates, and each plate was inoculated with 0.1 ml of virus sampleper plate. The cultures were incubated at 35° C., 5% CO₂ for 1 hr., andfed with 5 mL of medium per plate. After seven days, the plates werescanned microscopically for formation of VZV plaques (cytopathiceffects, CPE). P1 plates inoculated with VZV material from heated vials1-4 showed no detectable CPE. Plates inoculated with normal VZV had 90%CPE, (the uninoculated plate at every passage level had no CPE). Eachset of plates was washed with PBS, and the cells were harvested byaddition of 0.5 ml trypsin per plate and incubated 5 min. The cells fromeach grouping of five plates (i.e., five for isolate 1, five for isolate2 etc.) were removed and pooled. The pooled cells for each isolate werepelleted, and the supernatant was aspirated. Medium (25 mL) was thenadded to the pelleted cells to form a P1 suspension which was used toinoculate MRC-5 plates to form passage level 2:

Passage level 2, P2

Five 60 mm plate per isolate were each inoculated with 5 mL of P1 cellsuspension after aspirating medium from the MRC-5 cells. After 5 days,the plates were scanned microscopically. Plates from heated vials 1-3each had 1-3 visible plaques. Plates inoculated with heated vial number4 VZV showed no signs of CPE. The plates inoculated with unheated VZVhad 90% CPE. Each set of plates was washed with phosphate bufferedsaline and then treated with 0.5 mL trypsin. For each set, cells fromthree plates were pooled, pelleted and resuspended in 6 mL of freezingdiluent (EMEM, 15% DMSO, 10% fetal calf serum), aliquoted and stored inliquid nitrogen in 1 mL aliquots. The remaining two plates per set werepooled, pelleted and resuspended in medium (20 mL) to form a P2suspension which was used to inoculate MRC-5 plates to form passagelevel 3:

Passage level 3, P3

Four 60 mm plates per isolate were each inoculated with 5 mL of the P2isolate per plate after aspirating medium from the MRC-5 cells. Afterthree days, the plates were scanned microscopically. Plates from heatedvials 1-3 had 40%, 20% and 20% CPE respectively. Plates inoculated withheated vial number 4 VZV showed no signs of CPE. The plates inoculatedwith unheated VZV had 20% CPE. Each set of plates was washed withphosphate buffered saline and then treated with 0.5 mL trypsin. For eachset, all the cells were pooled using 5 mL media per plate, pelleted andresuspended in 6 mL of freezing diluent (EMEM, 15% DMSO, 10% fetal calfserum), aliquoted and stored in liquid nitrogen in 1 mL aliquots, toform the P3 stock used to inoculate cells for P4:

Passage level 4, P4

Two 150 cm² flasks were each inoculated with 1 mL of thawed P3 isolate(except for isolate 4 which was aborted due to non-appearance of any CPEup to P3). After 3 days flasks inoculated with heated isolates, 1-3 eachhad 70% CPE. Flasks inoculated with P3 of the unheated VZV isolate had40% CPE. Each flask was rinsed three times with phosphate bufferedsaline, aspirated, and the cells harvested by adding 5 mL trypsin toeach flask and incubating for 5 minutes at 35° C. Cells from each set offlasks were pelleted and resuspended in 30 mL of medium, to form the P4stocks used to inoculate cells for P5:

Passage level 5, P5

Sets of six 150 cm² flasks per set were each inoculated with 5 mL of P4suspension per flask. After 2 days, isolate 1-3 flasks each had 50-60%CPE while flasks inoculated with normal P4 VZV each had about 40% CPE.Each flask was washed three times with phosphate buffered saline andaspirated. VZV stabilizing solution was added to each flask (7 mL) andthe cells recovered by scraping, and then aliquoting 15 mL per 50 mLcentrifuge tube. Each tube was then sonicated for 5 minutes followed bycentrifugation at 2200 rpm for 10 minutes. The supernatants from eachset were pooled and aliquoted in 2.5 mL aliquots and frozen at -70° C.

The foregoing description of virus growth for each isolate and normalCR453 is summarized in Table

                  TABLE I                                                         ______________________________________                                        ISOLATION AND PROPAGATION OF THERMOSTABLE VZV                                 VARIANTS: # OF PLAQUES PER PLATE (PASSAGE 2) OR %                             CPE ON PLATE (FOR ALL OTHER PASSAGES):                                        ISO-        ISO-    ISO-                                                      LATE 1      LATE 2  LATE 3  ISOLATE 4                                                                             NORMAL 453                                ______________________________________                                        PASSAGE 1                                                                              0       0       0     0      90                                      5 × 60 mm                                                               plates @ 7                                                                    days                                                                          PASSAGE 2                                                                             1-3     1-3     1-3   0       90                                      5 × 60 mm                                                                       plaques plaques plaques                                               plates @ 4                                                                    days                                                                          PASSAGE 3                                                                             40      20      20    0       20                                      4 × 60 mm                                                               plates @ 3                                                                    days                                                                          PASSAGE 4                                                                             70      70      70    *       40                                      2 × 150 cm.sup.2                                                        flasks @ 3                                                                    days                                                                          PASSAGE 5                                                                             60      60      55    *       40                                      6 × 150 cm.sup.2                                                        flasks @ 2                                                                    days                                                                          ______________________________________                                         *Isolate was aborted at passage 3 due to absence of any detectable CPE.  

The growth of each heated isolate at each passage level appeared to besimilar. Virus stocks produced at Passage 5 for Isolates 1, 2 and 3along with normal CR453 were harvested and stored as sonicated,cell-free virus at -70° C. Passage 3 of Isolate 4 was aborted because ofno apparent cytopathic effect (CPE).

To determine the number of plaque-forming units for each stock, a plaqueassay was performed:

    ______________________________________                                        VIRUS STOCK          TITER                                                    ______________________________________                                        ISOLATE 1, (tVZV1)   86,200 pfu/ml                                            ISOLATE 2, (tVZV2)   69,000 pfu/ml                                            ISOLATE 3, (tVZV3)   10,840 pfu/ml                                            CONTROL 453          35,000 pfu/ml                                            ______________________________________                                    

For deposit with the ATCC, P5 virus of each tVZV isolate was expanded atpassage level 6 and frozen in 1 mL aliquots. tVZV1 was accorded ATCCnumber VR2437. tVZV2 was accorded ATCC number VR2438. tVZV3 was accordedATCC number VR 2439.

STABILITY ANALYSIS

For each isolate of virus stock, (tVZV1, tVZV2, tVZV3, and control,unheated CR453) a 1:20 dilution of the P5 material was made in astabilizing solution at 35° C. and incubated at 35° C. Samples weretaken from each diluted isolate at time intervals of 0 hr, 0.5 hr, 1.0hr, 1.5 hr, 2.0 hr, 2.5 hr, and 3.0 hr. Aliquots were frozen and storedat -70° C.

Samples from each time-point were then tested by plaque assay todetermine and compare stability. Six replicate stability assays wereperformed in this manner to determine the rates of decay for eachisolate and the control vaccine. The three tVZV isolates appear to havesimilar rates of decay (see Table 2 and FIG. 1). An average value of thesix stability assays shows there is no significant difference among theisolates (decay rate=50% per hr). However, the control vaccine CR453decayed at a rate of 79% per hr. In a lyophilized state, control vaccineloses viability at a rate of approximately 28% per day at 30 degrees.For lyophilized tVZV, this rate is predictably slower by approximately38% (50/79; i.e., only about 17% loss per day). The slopes of eachisolate showed a consistently lower rate of decay when compared to thecontrol vaccine for each stability assay.

                  TABLE 2                                                         ______________________________________                                        VARICELLA ISOLATE STABILITY EXPERIMENT                                        ESTIMATED FOLD DECREASE IN POTENCY PER HOUR (%)                               RUN   CONTROL    ISOLATE 1 ISOLATE 2                                                                              ISOLATE 3                                 ______________________________________                                        1     1.79 (79%) 1.40 (40%)                                                                              1.34 (34%)                                                                             1.70 (70%)                                2     1.76 (76%) 1.43 (43%)                                                                              1.36 (36%)                                                                             1.32 (32%)                                3     1.44 (44%) 1.24 (24%)                                                                              1.38 (38%)                                                                             1.28 (28%)                                4     1.74 (74%) 1.53 (53%)                                                                              1.51 (51%)                                                                             1.68 (68%)                                5     1.94 (94%) 1.52 (52%)                                                                              1.44 (44%)                                                                             1.80 (80%)                                6     2.18 (118%)                                                                              1.80 (80%)                                                                              1.69 (69%)                                                                             1.58 (58%)                                Pooled                                                                              1.79 (79%) 1.49 (49%)                                                                              1.46 (46%)                                                                             1.55 (55%)                                Average                    1.50 (50%)                                         ______________________________________                                         Note: There is a statistically significant difference in slopes between       the control and heated isolates, while there is no difference among the       heated isolates (P < 0.01).                                              

EXAMPLE 3

Assay For VZV Yield Determination

The infectivity titers of varicella zoster virus (VZV) preparations wereestimated using the agarose-overlay or liquid overlay proceduredescribed by Krah et at., (J. Virol. Methods, 1990, 27:319-326). Theassay for the liquid overlay and the agarose-overlay method is performedas follows:

MRC-5 cells are seeded in 60-mm tissue culture plates, at 6×10⁵ cells in5 mL volumes of BME (Basal Medium Eagle with Hanks'balanced saltssolution) with 1000 mg/L galactose, 50 μg/mL neomycin, 2mM L-glutamine,and are incubated at 35° C. in a 5% CO₂ atmosphere. After incubation for24-48 hours, the cells reach 50-80% confluency. The growth medium isremoved by aspiration, and cells are infected with 100 μl of VZVsolution diluted in appropriate virus diluent, such as SPGA buffer, orliquid maintenance medium (LMM). SPGA buffer contains 7.5% (w/v)sucrose, 11 mM potassium phosphate, 0.1% (w/v) sodium glutamate and 1%human serum albumin. Virus is allowed to attach for >1 hour at 35° C. ina 5% CO₂ atmosphere. The VZV-infected cell cultures are then overlaidwith 5 mL agarose overlay medium (AOM) or liquid maintenance medium(LMM). Agarose overlay medium is a mixture of two solutions, liquidoverlay medium (LOM) and agarose solution. LOM contains minimalessential medium with Earle's salts (MEM), 2% heat-inactivated fetalcalf serum, 50 μg/mL neomycin sulfate and 2 mM L-glutamine. Agarosesolution is prepared by heating 4.5 g of low gelling temperature agarosein 100 mL MEM for 15 min at 121° C. and allowing the solution to cool to45° C. AOM is prepared by mixing one volume of agarose solution with 4volumes of a 1.25×concentrate of LOM at 45° C., (for the liquid overlaymethod, a 1× concentration of LOM is used and need not be heated orcooled). The plates are cooled to 23-25° C. to permit the AOM tosolidify. The cultures are incubated to allow plaque development. After6-7 days, plates which received AOM are overlaid with 5 mL ofphosphate-buffered saline (PBS) and rimmed with a glass Pasteur pipetteto loosen and remove the agarose. Medium is aspirated from plates whichreceived LMM. Plaques are visualized by staining cells with a solutionof 0.2% (w/v) Coomassie Blue R-250 in ethanol-1% acetic acid. Plaquecounts are the average of 4-5 replicate plates and expressed asplaque-forming units per mL (PFU/mL).

EXAMPLE 4

Quantitative Dot-Blot Assay Of Varicella-Zoster

The procedure described herein allows quantitation of VZV antigen. It isaccomplished by measuring actual counts per minute of radioactivity inreacted dot-blots rather than using X-ray film. This procedure isintended for estimating total VZV antigen in clarified dispensed VZVvaccine bulk samples.

Materials and Reagents

A. Equipment

1. Polystyrene snap-lock boxes approximately 8"×12"×11/2".

2. Rocker platform (Bellco Glass Co., Model 6-044).

3. Nitrocellulose. (ProMega Biotec, 0.45 μl pore size, 15×15 cm).

4. 4° C. Refrigerator or Cold Room.

5. Pipetters 0-100 μl dispensing with disposable tips.

6. Micro Centrifuge Tubes, disposable, polypropylene 1.5 ml capacity.

7. Gamma Counter suitable for counting ¹²⁵ I.

8. Disposable test tubes for use in Gamma Counter.

B. Reagents

1. Phosphate-buffered saline.

2. Sodium Dodecyl Sulfate (SDS) Bio-Rad Catalog #161-0302.

3. Nonidet NP-40 (Sigma No. N6507).

4. Fetal Calf Serum (FCS).

5. Albumin, Bovine Fraction V (BSA) Calbiochem #12659.

6. Sodium Azide (Sigma).

7. ¹²⁵ I Protein A 0.1 mCi/ml (Amersham). To be used within one monthdate of product.

8. VZV Reference Antigen (VZV Lot 851) stored at -70° C.

9. VZV Positive Antiserum (O.L.I. high-titered zoster serum, labeledHSV143-80 O.I., 8/20/80). Human. 10. Four roller bottles of confluentMRC-5 cells.

11. Whatman 46 cm×57 cm -- 3 mm paper (blotting paper) (catalog#3030917).

12. Triton X-100 (TX-100) ('Sigma No. T6878).

13. VZV Negative Control Antigen.

C. Preparation of Reagents

1. Quench Buffer (Q Buffer):

a. 3L RCM8

b. 6 gm SDS

c. 0.6 gm Sodium azide

d. 70 gm BSA

e. 30 ml Nonidet NP-40

2. Quench buffer with 1% FCS (blocking buffer)

a. 50 ml quench buffer+0.5 ml FCS

3. RCM8 with 0.05% Triton X-100

4. Antisera: O.L.I. Human Zoster Serum

a. dilute 1:2500 in 50 ml Q buffer

5. Sonicate MRC-5 cell extracts: Scrape 4 roller bottles with 5 mlRCMS/bottle, then rinse all 4 bottles with 10 ml RCMS; pool all. Cool to4° C., sonicate 3 min and bring to 40 cc with RCM8.

6. ¹²⁵ I Protein A Solution: Dilute 125 μl+50 lI Q buffer (0.25 μCi/ml).

Procedure

A. Day 1

1. Prepare sonicated MRC-5 cell extract (cell solution) and place insnap-lock box, along with 2 nitrocellulose sheets 20 cm×40 cm (blockingsheet)!.

2. Rock at 4° C. approximately 3 hours.

3. Add 50 ml Quench buffer and rock an additional hour.

4. Make antibody dilution, remove blocking sheets from cell solution andplace one blocking sheet in antibody solution. Rock overnight at 4° C.

NOTE: Step 1 through 4 can be done several days before assay isperformed; store blocked antisera at 4° C. (remove blocking sheets anddiscard sheets after overnight rock in this case).

B. Day 2

5. Add Triton X-100 to 0.05% in undiluted test samples. Draw grid onnitrocellulose sheet to account for all samples (Attachment 111). Dilutereference vaccine (851) and negative control 1:8 in RCM8 containing0.05% TX-100. Serially dilute 2-fold through 1:64. Test samples arediluted in same manner; however, only 1:16 and 1:32 dilutions are used.Antigens are spotted in duplicate, 5 μl per dilution, on thenitrocellulose grid. Let dry 15 min at room temperature.

6. Place nitrocellulose test sheet in Q buffer with 1% FCS (immerseslowly and evenly) and rock at 4° C. for 1.5 to 18 hours.

7. Remove blocking sheets from antibody solution, place testnitrocellulose sheet into antibody solution and rock overnight at 4° C.

C. Day 3

8. Wash nitrocellulose with Quench buffer on rocker approximately 2hours, changing buffer every 20 min. Use about 50 ml/wash.

9. Drain and add 50 ml ¹²⁵ I! protein A in Q buffer to test sheets androck 2 hrs on rocker. Dispose of all radioactive materials inappropriate radiation waste containers.

10. Wash test sheet with Q buffer approximately 1.5 hrs, change bufferevery 20 min. Dispose of first 4 washes in containers for radiation.

11. Blot test sheet with blotting paper.

12. Cut test sheets according to grid, and place each individualdilution blot into a labelled test tube.

13. Count each tube 1 min using ¹²⁵ I Gamma Counter.

14. Calculation of Results: A standard antigen has 26 units of VZVantigen per ml. At 1:8, 1:16, 1:32, and 1:64 dilutions, it has 3.25,1.63, 0.81, and 0.40 traits of antigen per ml. A standard curve isconstructed for each run of the assay by plotting mean c.p.m. obtainedat each known concentration of antigen. The antigen concentration in thetest samples is obtained by finding the antigen level on the Abscissacorresponding to the mean c.p.m. at each test dilution. The finalantigen estimate is the mean of the values obtained at each testdilution. Antigen values for undiluted test samples are calculated byappropriate correction of dilution.

MRC-5 antigen is included in each test as a control in the procedure. Inpractice, only .low c.p.m. have been detected in MRC-5 blots and nocorrections of test antigen c.p.m. have been made.

EXAMPLE 5

ELISA For Quantitation Of VZV Antigen

A rapid VZV antigen ELISA permits measurement of VZV antigen amounts topermit monitoring of virus growth during manufacture of live varicellavaccine. Additionally, this test can be used to estimate VZV antigenamounts in clarified, sonicated vaccine bulks, and potentially tomeasure antigen in filled lyophilized vaccine vials. Briefly, this assayis conducted by incubation of VZV antigen from test samples withanti-VZV serum in solution. Remaining free antibody is allowed to bindto VZV antigen mobilized on ELISA microtiter plates. The amount ofantibody capable of binding to the plates is inversely proportional tothe amount of antigen in the test sample. Antibody binding to the platesis quantitated by reaction with an enzyme-linked anti-human antibody andappropriate substrate to provide a colored product which is quantitatedspectrophotometrically.

The VZV antigen ELISA and the VZV plaque assays should generally providecorrelative data, but it should be borne in mind that the VZV antigenassay detects non-viable as well as viable VZV. Since the immuneresponse generated by killed VZV has not been shown to be as effectiveas the response to live attenuated virus, the plaque assay is thecritical assay for determination of viral inoculum dose for VZVvaccines. However, the antigen assay is also valuable in that itprovides a measure of the total antigen load being administered to a VZVvaccine recipient.

Test Procedure

1. ELISA plates are coated with glycoproteins (gps) from VZV-infected oruninfected MRC-5 cells, and are overcoated with 1% bovine serum albuminfraction V, #A-9647, Sigma!, 0.1% NAN3) to reduce non-specificadsorption of antibodies to the plates. Alternating rows are coated withVZV or control antigen (i.e., rows A, C, E, and G receive VZV gp androws B, D, F, and H receive uninfected MRC-5 gp antigen).

2. Clarified (3250 g-min) test antigen is diluted in stabilizer in 12×75mm tubes or microtubes. A standard virus antigen preparation (26units/mL VZV antigen by dot blot assay) is diluted 1:10 and thenserially 1:1.25-fold to provide antigen concentrations of 2.6, 2.1, 1.7,1.3, 1.1, 0.9 units/mL. Additional dilutions may be included to provide0.7 and 0.5 units/mL of antigen. This dilution series is used togenerate a standard curve for the measurement of antigen amounts in testsamples.

3. A human anti-VZV serum is diluted in stabilizer to 2 times the finaldesired dilution.

4. Three hundred ml volumes of diluted antigen are dispensed intomicrotubes, mixed with 300 ml diluted anti-VZV serum and incubated at35° C. for 15-22 min. A control includes human anti-VZV and diluent (noantigen).

5. Aliquots of 100 ml from each serum-antigen mixture are added to 2replicate VZV glycoprotein (VZV gp) coated wells and 2 MRC-5 gp coatedwells (4 wells per sample) (e.g., sample 1 in column 1, rows A, B, C,and D; sample 2 in column 2, rows A, B, C, and D; etc.).

6. Plates are incubated for 15+1 minute at 35° C. to allow free antibody(not complexed to antigen in solution) to bind to virus antigenimmobilized on the plates.

7. Unbound antibody is removed by washing and wells receive an alkalinephosphatase conjugated goat anti-human IgG to detect bound humanantibody.

8. After incubation for 15+1 minute at 35° C., unbound conjugate isremoved by washing. Bound conjugate is detected by incubation for 15 minat 35° C. with p-nitrophenyl phosphate substrate dissolved indiethanolamine buffer.

9. After termination of the substrate reaction by addition of 50 ml/well3 M NaOH, color development (OD at 405 nm) is quantitated using amicroplate spectrophotometer.

1. Test Calculations and Interpretation

Respective replicate OD values for the replicate VZV and MRC-5 coatedwells are averaged. Experience has shown the MRC-5 OD to be consistentbetween different samples and dilutions. Therefore, the MRC-5 values forthe entire plate are averaged and used to correct for non-specificbinding of the primary antibody or conjugate to uninfected cellextracts. The averaged MRC-5 OD is subtracted from the respectiveaveraged VZV ODs to provide VZV-specific OD (ΔOD) values.

2. Generation of a standard curve for measurement of antigen amounts

The standard curve ΔOD values are plotted against the known antigenconcentrations (units VZV/mL). The data are entered into an appropriategraphics program (e.g., Cricket Graph version 1.3, Cricket Software,Malvern, Pa.), the linear portion of the curve is identified (mustinclude at least 4 points), and the "line fit formula" (y=a+bx) isobtained.

3. Calculation of antigen amounts of test samples

Values for a and b are given by the line-fit formula, and y (ΔOD) isknown. The remaining unknown value, x, representing the units/mLantigen, can then be calculated, and corrected by the sample dilution toobtain the antigen concentration of the undiluted sample. The reportedantigen concentration is that obtained with the least diluted sampleproviding a ΔOD value within the linear portion of the standard curve.

What is claimed is:
 1. A process for preparing a live attenuatedthermostable varicella zoster virus (tVZV) which comprises:a) heating alyophilized or liquid preparation of live attenuated VZV under highlyinactivating conditions; and b) selecting by culturing residual liveattenuated VZV.
 2. The live attenuated tVZV obtained according to theprocess of claim
 1. 3. A thermostable varicella zoster virus having arate of inactivation equal to about 50% per hour at 35° C. in a liquidmedium.
 4. A method of using the tVZV of claim 2 to make a VZV vaccinewhich comprises combining the tVZV with a pharmaceutically acceptablecarrier and providing sufficient tVZV such that a minimum of about 1000plaque forming units of the virus is available on the date ofvaccination in the pharmaceutically acceptable carrier.
 5. A method ofusing the tVZV of claim 3 to make a VZV vaccine which comprisescombining the tVZV with a pharmaceutically acceptable carrier andproviding sufficient tVZV such that a minimum of about 1000 plaqueforming units of the virus is available on the date of vaccination inthe pharmaceutically acceptable carrier.
 6. A vaccine comprising thetVZV of claim
 2. 7. A vaccine comprising the tVZV of claim
 3. 8. Athermostable virus selected from the group consisting of ATCC VR2437,ATCC VR2438, and ATCC VR2439.
 9. A method of obtaining a tVZV variantwhich has a rate of inactivation at 35° C. in a liquid medium which isabout 38% slower than the live attenuated VZV population from which thetVZV was selected, which comprises:a) heating a lyophilized or liquidpreparation of live attenuated VZV for about 5-19 days at a temperatureof 25°-75° C.; and b) selecting by culturing residual live VZV, thusobtaining a tVZV variant which has a rate of inactivation at 35° C. in aliquid medium which is about 38% slower than the VZV population fromwhich the tVZV was selected.
 10. A vaccine comprising the tVZV obtainedaccording to the method of claim
 9. 11. A multivalent vaccine comprisingthe tVZV obtained according to the method of claim
 9. 12. The method ofclaim 1 wherein the selected virus is cultured through between one andsix cell culture passages.
 13. A method for producing a morethermostable VZV (tVZV) than is available in a given live attenuated VZVisolate which comprises:a) heating a lyophilized or liquid preparationof live attenuated VZV for about 5-19 days at a temperature of 25°-75°C.; and b) selecting by culturing residual live VZV, thus producing amore thermostable VZV (tVZV) than is available in the given liveattenuated VZV isolate.